Team:Grenoble-Alpes/phage lysis

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DNA EXTRACTION

The first main step in our system is the lysis of bacteria by bacteriophages. This step is decisive for all the rest of the system. In fact this step allows the release of DNA in the medium by lysing the specific bacteria. It is this specific DNA that will be extracted, digested and detected by our plasmid probes. So if the DNA released hybridize to the probes, it gives two essential information:

  • Which  bacterial species are present in the patient sample.
  • What are the best bacteriophages capable of infecting this bacteria, and thus, treating the patient.


    • About bacteriophages

    Titre


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    Figure 1: Result of alignment of the target sequence of the detection of bacterial lysis in PAO1.

    This result isn’t problematic as the specificity will be provided by the phage. Indeed, a phage is specific of a bacterial strain, so a screening of phages will be done before their use in the device. Finally, the target found in ProC gene (822bp) from PAO1 strain (GenBank : AAG03782.1) is located in PA0393 locus. The target is located between nucleotide 766 and nucleotide 802.

    5’- CCCTGAACGCCGCCAGCCAGCGCTCCGCCGAGCTGG-3’

    Figure 2: Target of lytic biologically selected PAO1 lysis.

    As for the enzyme surrounding the sequence on both sides it is HaeIII.

    Figure 3: Recognition Site of the HaeIII Enzyme
    Figure 4: Target chosen surrounded by HaeIII, blunt ends restriction enzyme.

    All this work has been carried out in order to select the target after extracting Pyo's DNA. Indeed, after the extraction, HaeIII digestion during 15 minutes at 37 ° C in CutSmart Buffer will be performed. The target is now ready to be detected and the next step, the detector activation, can occur.


    Target that characterizes a marker of resistance of Pyo


    When selecting the gene fragment to target markers of resistance, the strategy was the same. Indeed, we had to choose a fragment with these 3 characteristics but the feature here is to find a short fragment with more than 1 mutation.

    Pyo has developed many resistance mechanisms during evolution. The most common mechanisms are multiple mutations leading to AmpC overexpression (ceftazidime), oprD inactivation (meropenem), modification of type II topoisomerases, as well as overexpression of the efflux pump (ciprofloxacin and meropenem) [4].

    By doing some reading, we selected the gene that mutated the most and looked for common mutations on it. The selected gene was gyrA (GenBank: AAG06556.1) gene. It encodes for the DNA gyrase subunit A (topoisomerase II) and is on the PA3168 genome PA3168 locus. The gyrase DNA mutation leads to resistance to fluoroquinolones [5] [6]. Fluoroquinolones are antibiotics acting on DNA gyrase, it prevents replication of bacterial DNA and thus bacterial proliferation.

    We noticed in several articles explaining the mutations of the gyrA gene of Pyo clinical strains, that some were redundant. First, at position 83 of the gyrA gene, threonine becomes an isoleucine (Thr83Ile) [5] [6] [7] [11]. Moreover, in position 87 an aspartate becomes an asparagine (Asp87Asn) [5] [7] [8]. So we decided to work on these two mutations. The advantage is that they are close in the gyrA gene (12 nucleotides between them), this is an important point because the detection target sequence must not exceed 50 nucleotides. Once the location was found, the gene mutated was inserted in NebCutter to see natural restriction sites.

    Finally, a sequence alignment in BLAST was performed to ensure that the fragment was only found in Pyo. The parameters of the BLAST are : Excluded “Pseudomonas aeruginosa group” and “ Highly similar sequences, megablast”. The result (Fig.5) shows 4 alignment results with 100% homology. For two of them, the result is normal because it corresponds to the gene coding for the subunit A of DNA gyrase (gyrA). For the 2 others, Pseudomonas Putida is found in most soils and aquatic habitats where there is oxygen. P.P is able to degrade organic solvents such as toluene, it is not found in clinical cases. This result isn’t problematic as the specificity will be provided by the phage.

    Indeed, we used HER18 phage that is it specific against PAO1 strain (taxonomy : txid280701). In addition, as previously said, the specificity will be provided by the phage. Indeed, a phage is specific for a bacterial strain, so a selection will have been made before using the engineering system. With all these elements, we can ignore this homology.